Low density lipoprotein receptor-related protein-1 (LRP-1) is a transmembrane receptor that mediates the endocytosis of over forty distinct ligands and other plasma membrane proteins. LRP-1 also regulates cell-signaling by two distinct mechanisms. Ligands that bind directly to LRP-1, including tissue-type plasminogen activator, activated 12-macroglobulin, and matrix metalloprotease-9, activate cell-signaling pathways leading to factors such as ERK/MAP kinase, Akt and possibly Src family kinases and NF-kB. LRP-1 also regulates cell- signaling indirectly by controlling cell-surface levels of other receptors, including the urokinase receptor (uPAR) and tumor necrosis factor receptor-1 (TNFR1). Recent studies from this and other laboratories have provided novel mechanistic links between LRP-1-initiated cell-signaling and inflammation, cell survival, and atherosclerosis. The major goal of this research proposal is to elucidate the function of LRP-1 in cell-signaling and to understand how LRP-1 regulates inflammation in vivo. Preliminary results support our major hypothesis that LRP-1 suppresses inflammation by its effects on cell-signaling as both a transmembrane and shed/soluble receptor. Three aims are proposed. These aims are presented in a systematic manner to show how our planned research moves from discovery (Aim 1) to assessment of molecular mechanism (Aim 2) and finally to in vivo model systems (Aim 3). In Aim 1, we will apply our novel ectodomain shaving protocols, the recently described PROTOMAP platform, and available LC-MS technology to assess the ability of LRP-1 to regulate the plasma membrane proteome. Pilot studies applying our proposed methodology have already revealed novel plasma membrane proteins that may be regulated by LRP-1 and that may contribute to the effects of LRP-1 on cell physiology. In Aim 2, studies are proposed to characterize how LRP-1 indirectly regulates cell-signaling downstream of TNFR1 and uPAR. We also will work to develop a model regarding how the cell integrates simultaneous signals received directly from LRP-1, due to ligand-binding, and from receptors that are indirectly regulated by LRP-1. We hypothesize that the predominant pathway will reflect the availability of ligands for LRP-1 and for the indirectly regulated receptors in the cellular microenvironment. Finally, in Aim 3, we will test the hypothesis that LRP-1 is anti-inflammatory in vivo due to its ability to regulate cell-surface TNFR1. Lipo- polysaccharide challenge experiments will be conducted in mice in which LRP-1 is conditionally deleted in macrophages and in control mice in the equivalent genetic background. Additional experiments are planned to test the activity of shed LRP-1 as an anti-inflammatory agent in vivo. Overall, these studies have the potential to further our understanding of diverse forms of pathophysiology in which inflammation plays a role, including thrombosis and atherogenesis.